Biological systems universally employ cascades of binding or catalytic events to transmit information. The central players in these cascades are proteins with binding sites for "input" ligands that change binding or catalysis at "outplt" sites. Allosteric mechanisms serve as the switch by which an input signal is converted to an output signal-most commonly through conformational changes or coupled binding/folding. This pivotal signalling process is poorly understood at the atomic level, especially for multi-domain transcription factors. We propose detailed examination of this switching pathway in the lactose repressor protein (LacI). LacI inhibits transcription of the lac metabolic enzymes by binding tightly to specific operator sites within the E. coli genome. When LacI binds inducer sugar, DNA binding is diminished and the metabolic genes are transcribed. Recent crystallographic structures for various liganded forms of LacI provide snapshots of the conformational states of LacI, but give no direct information on the molecular pathway(s) between these states. A unique opportunity exists to couple recent structural information and the vast phenotypic data on LacI mutants with detailed biochemical and biophysical characterization methods developed in our laboratory to explore allosteric signal transmission in LacI at the atomic level. Ligand binding information must flow through the structure of LacI between the widely separated inducer and DNA binding sites. Structurally, this linkage is provided by the hinge helix, which is folded only in the operator-bound form of LacI. Although the end states for the LacI allosteric change are known, the molecular mechanism of signal propagation remains unknown. This proposal is designed to elucidate the structural changes within LacI in response to DNA and inducer binding and to establish the allosteric pathway for this multi-domain transcription factor. The key hypotheses to be explored are: (1) DNA sequence influences binding and allostery through effects on hinge helix folding, (2) differences between inducer and anti-inducer ligands derive from their differential effect on hinge helix folding, and (3) specific amino acid changes can disrupt the allosteric pathway and block communication between the inducer and operator sites. To examine the local structures altered in the LacI allosteric mechanism, hydrogen exchange techniques will be added to our experimental repertoire of thermodynamic, chemical, and genetic methods. With this addition, all tools are in place to uniquely detail the allosteric structural changes of a genetic regulatory protein.